Polyglucosan bodies in medullary catecholaminergic neurones in SUDEP

Polyglucosan bodies have been reported in the context of hypoxic-ischaemic perinatal brain injury, mainly in the pallidum but with rare reports in brainstem neurones. We report a case of a five year old boy with cerebral palsy, complex neurology and epilepsy with a sudden nocturnal death. At post-mortem long-standing bilateral necrosis of basal ganglia and hippocampal atrophy was identified in keeping with hypoxic-ischaemic perinatal injury. In addition numerous polyglucosan bodies, which were PAS, p62 and ubiquitin positive, were noted in brainstem neurones and dendrites, primarily involving the ventrolateral and dorsomedial medulla. Immunohistochemistry confirmed relative preservation of medullary neuronal populations in the reticular formation, including catecholaminergic (tyrosine hydroxylase, TH), serotonergic (tryptophan hydroxylase) and neurokinin1 receptor/somatostatin positive neurones. The polyglucosan bodies predominated in catecholaminergic neurones which could indicate their selective vulnerability and a functional deficiency, which during a critical peri-ictal period contributed to the sudden death in epilepsy

Review: Neuropathology findings in autonomic brain regions in SUDEP and future research directions

Autonomic dysfunction is implicated from clinical, neuroimaging and experimental studies in sudden and unexpected death in epilepsy (SUDEP). Neuropathological analysis in SUDEP series enable exploration of acquired, seizure-related cellular adaptations in autonomic and brainstem autonomic centres of relevance to dysfunction in the peri-ictal period. Alterations in SUDEP compared to control groups have been identified in the ventrolateral medulla, amygdala, hippocampus and central autonomic regions. These involve neuropeptidergic, serotonergic and adenosine systems, as well as specific regional astroglial and microglial populations, as potential neuronal modulators, orchestrating autonomic dysfunction. Future research studies need to extend to clinically and genetically characterized epilepsies, to explore if common or distinct pathways of autonomic dysfunction mediate SUDEP. The ultimate objective of SUDEP research is the identification of disease biomarkers for at risk patients, to improve post-mortem recognition and disease categorisation, but ultimately, for exposing potential treatment targets of pharmacologically modifiable and reversible cellular alterations

Transcriptional regulation and downstream effectors of peripheral nerve regeneration

Unlike the central nervous system, robust axon regeneration after peripheral nerve injury is driven by transcriptional activation of a regeneration programme. Transcription factors form a vital link in the chain of regeneration, converting injuryinduced stress signals into increased expression of a wide range of downstream effector molecules like neuropeptides, neurotrophic factors, adhesion molecules and cytoskeletal adaptor proteins. To gain insight into the in vivo function of some of these regeneration associated molecules, we examined the effects of global or celltype specific, and single and combined deletions of transcription factors - STAT3, c- Jun and C/EBPδ, and effector molecules - CAP23 and integrin β1, on axonal regeneration after facial nerve axotomy. The facial nerve axotomy model is a wellestablished experimental paradigm, providing insights into molecular signals that determine axonal regeneration, target re-innervation and neuronal cell death. Neuronal STAT3 deletion (STAT3ΔS) caused profound and persistent defects in regeneration and functional recovery after mild (crush) and severe (cut) nerve injuries. Axotomised STAT3-deficient motoneurons appeared shrunk by 50-60% in size but displayed no cell death. There was also a severe reduction in microglial activation and recruitment of lymphocytes, in the expression of regenerationassociated molecules CD44, α7β1 integrin, in the nuclear translocation of ATF3, and in perineuronal sprouting of the CGRP+ and galanin+ facial motoneurons. Deletion of STAT3 in Schwann cells produced no apparent deficiencies in regeneration. As with neuronal STAT3 deletion, neuronal deletion of c-Jun (c-JunΔS) (Raivich et al, 2004; Ruff et al, 2012) abolishes most of the cell body response, neuronal cell death, and re-innervation after axotomy. Deletion of c-Jun in peripheral nerve Schwann cells (c-JunΔP0) had a very different effect. Neuronal cell death was increased by 2- 3 fold, even though most of the cell body response was not affected. Axonal regeneration was reduced, but most of the defect in target re-innervation and functional recovery appeared to be due to excessive neuronal cell death. Mutants lacking both neuronal c-jun and STAT3 did not show more exacerbated regeneration defects than the single deletions, suggesting that deletion of either 4 transcription factor – c-Jun or STAT3 – will produce a saturating effect on the regeneration-deficient phenotype. Expression of c-jun itself was not affected in the neuronal STAT3 null mice, and vice versa, confirming the above hypothesis. Double deletion of c-jun in both neurons and in Schwann cells, blocked neuronal cell death seen in Schwann cell c-Jun deficient mutants, and completely obliterated target re-innervation after facial axotomy, suppressing even the mild 20% regeneration seen in the neuronal c-jun null mutants. Since both neuronal and Schwann cell c-Jun are phosphorylated at its N-terminus following nerve injury, we also explored the effects of c-Jun phosphorylation. Global replacement of all 4 Nterminal c-Jun phosphorylation sites (Ser63&73, Thr91&93) with alanines (c-jun4A) produced a significant increase (1.8x) in neuronal cell death, an approximate 40% reduction in target re-innervation and delayed functional recovery. This phenotype was more alike that observed with Schwann cell rather than neuronal c-Jun deletion. Global C/EBPδ deletion reduced axotomy induced neuronal cell death, and had moderate effects on microglial activation and axonal sprouting. Combined deletion of C/EBPδ and STAT3 did not exacerbate the defect in regeneration seen with STAT3 deletion alone, but seemed to speed up functional recovery. Deletion of neuronal CAP23, a downstream effector gene, led to impaired target reinnervation, reduced galanin+ perineuronal sprouting and early microglial function. Neuron-specific deletion of beta 1 integrin, another effector molecule, resulted in a 2.5-fold increase in neuronal cell death, a commensurate 60% reduction in target reinnervation and transiently delayed functional recovery. This phenotype was similar to c-junΔP0 and c-jun4A mutants. In summary, there appears to be two neuronal sub-populations which are controlled by different aspects of the regenerative programme A) one dependent on ‘intrinsic’ activation of transcriptional master switches like c-jun and STAT3 in neurons and B) the other on ‘extrinsic’ post-traumatic trophic signalling elicited by the Jun-expressing and N-terminal phosphorylation-dependent Schwann cells. c-Jun phosphorylation and neuronal β1 integrin appear to be critical co-factors in the signalling response elicited by the Schwann cells

Role of transcription factors in peripheral nerve regeneration.

Following axotomy, the activation of multiple intracellular signaling cascades causes the expression of a cocktail of regeneration-associated transcription factors which interact with each other to determine the fate of the injured neurons. The nerve injury response is channeled through manifold and parallel pathways, integrating diverse inputs, and controlling a complex transcriptional output. Transcription factors form a vital link in the chain of regeneration, converting injury-induced stress signals into downstream protein expression via gene regulation. They can regulate the intrinsic ability of axons to grow, by controlling expression of whole cassettes of gene targets. In this review, we have investigated the functional roles of a number of different transcription factors - c-Jun, activating transcription factor 3, cAMP response element binding protein, signal transducer, and activator of transcription-3, CCAAT/enhancer binding proteins β and δ, Oct-6, Sox11, p53, nuclear factor kappa-light-chain-enhancer of activated B cell, and ELK3 - in peripheral nerve regeneration. Studies involving use of conditional mutants, microarrays, promoter region mapping, and different injury paradigms, have enabled us to understand their distinct as well as overlapping roles in achieving anatomical and functional regeneration after peripheral nerve injury

Medullary tyrosine hydroxylase catecholaminergic neuronal populations in sudden unexpected death in epilepsy

Sudden unexpected death in epilepsy (SUDEP) is mechanistically complex and one probable cause is seizure‐related respiratory dysfunction. Medullary respiratory regulatory nuclei include the pre‐Bötzinger complex (pre‐BötC) in the ventrolateral medulla (VLM), the medullary raphé nuclei (MR) and nucleus of solitary tract in the dorsomedial medulla (DMM). The region of the VLM also contains intermingled tyrosine hydroxylase (TH) catecholaminergic neurones which directly project to the pre‐BötC and regulate breathing under hypoxic conditions and our aim was to evaluate these neurones in SUDEP cases. In post‐mortem cases from three groups [SUDEP (18), epilepsy controls (8) and non‐epilepsy controls (16)] serial sections of medulla (obex + 2 to + 13 mm) were immunolabeled for TH. Three regions of interest (ROI) were outlined (VLM, DMM and MR) and TH‐immunoreactive (TH‐IR) neurones were evaluated using automated detection for overall labeling index (neurones and processes) and neuronal densities and compared between groups and relative to obex level. C‐fos immunoreactivity was also semi‐quantitatively evaluated in these regions. We found no significant difference in the density of TH‐IR neurones or labeling index between the groups in all regions. Significantly more TH‐IR neurones were present in the DMM region than VLM in non‐epilepsy cases only (P < 0.01). Regional variations in TH‐IR neurones with obex level were seen in all groups except SUDEP. We also identified occasional TH neurones in the MR region in all groups. There was significantly less c‐fos labeling in the VLM and MR in SUDEP than non‐epilepsy controls but no difference with epilepsy controls. In conclusion, in this series we found no evidence for alteration of total medullary TH‐IR neuronal numbers in SUDEP but noted some differences in their relative distribution in the medulla and c‐fos neurones compared to control groups which may be relevant to the mechanism of death

Characterisation of medullary astrocytic populations in respiratory nuclei and alterations in sudden unexpected death in epilepsy

Central failure of respiration during a seizure is one possible mechanism for sudden unexpected death in epilepsy (SUDEP). Neuroimaging studies indicate volume loss in the medulla in SUDEP and a post mortem study has shown reduction in neuromodulatory neuropeptidergic and monoaminergic neurones in medullary respiratory nuclear groups. Specialised glial cells identified in the medulla are considered essential for normal respiratory regulation including astrocytes with pacemaker properties in the pre-Botzinger complex and populations of subpial and perivascular astrocytes, sensitive to increased pCO2, that excite respiratory neurones. Our aim was to explore niches of medullary astrocytes in SUDEP cases compared to controls. In 48 brainstems from three groups, SUDEP (20), epilepsy controls (10) and non-epilepsy controls (18), sections through the medulla were labelled for GFAP, vimentin and functional markers, astrocytic gap junction protein connexin43 (Cx43) and adenosine A1 receptor (A1R). Regions including the ventro-lateral medulla (VLM; for the pre-Bötzinger complex), Median Raphe (MR) and lateral medullary subpial layer (MSPL) were quantified using image analysis for glial cell populations and compared between groups. Findings included morphologically and regionally distinct vimentin/Cx34-positive glial cells in the VLM and MR in close proximity to neurones. We noted a reduction of vimentin-positive glia in the VLM and MSPL and Cx43 glia in the MR in SUDEP cases compared to control groups (p < 0.05-0.005). In addition, we identified vimentin, Cx43 and A1R positive glial cells in the MSPL region which likely correspond to chemosensory glia identified experimentally. In conclusion, altered medullary glial cell populations could contribute to impaired respiratory regulatory capacity and vulnerability to SUDEP and warrant further investigation

Neuropeptide depletion in the amygdala in sudden unexpected death in epilepsy: A postmortem study

OBJECTIVE: Sudden unexpected death in epilepsy (SUDEP) is typically unwitnessed but can be preceded by seizures in the period prior to death. Peri-ictal respiratory dysfunction is a likely mechanism for some SUDEP, and central apnea has been shown following amygdala stimulation. The amygdala is enriched in neuropeptides that modulate neuronal activity and can be transiently depleted following seizures. In a postmortem SUDEP series, we sought to investigate alterations of neuropeptidergic networks in the amygdala, including cases with recent poor seizure control. METHODS: In 15 SUDEP cases, 12 epilepsy controls, and 10 nonepilepsy controls, we quantified the labeling index (LI) for galanin, neuropeptide Y (NPY), and somatostatin (SST) in the lateral, basal, and accessory basal nuclei and periamygdala cortex with whole slide scanning image analysis. Within the SUDEP group, seven had recent generalized seizures with recovery 24 hours prior to death (SUDEP-R). RESULTS: Galanin, NPY, and SST LIs were significantly lower in all amygdala regions in SUDEP cases compared to epilepsy controls (P < .05 to P < .0005), and galanin LI was lower in the lateral nucleus compared to nonepilepsy controls (P < .05). There was no difference in the LI in the SUDEP-R group compared to other SUDEP. Higher LI was noted in epilepsy controls than nonepilepsy controls; this was significant for NPY in lateral and basal nuclei (P < .005 and P < .05). SIGNIFICANCE: A reduction in galanin in the lateral nucleus in SUDEP could represent acute depletion, relevant to postictal amygdala dysfunction. In addition, increased amygdala neuropeptides in epilepsy controls support their seizure-induced modulation, which is relatively deficient in SUDEP; this could represent a vulnerability factor for amygdala dysfunction in the postictal period

MRI and pathology correlations in the medulla in SUDEP: A post-mortem study

Aims: Sudden unexpected death in epilepsy (SUDEP) likely arises as a result of autonomic dysfunction around the time of a seizure. In vivo MRI studies report volume reduction in the medulla and other brainstem autonomic regions. Our aim, in a pathology series, is to correlate regional quantitative features on 9.4T MRI with pathology measures in medullary regions. Methods: Forty‐seven medullae from 18 SUDEP, 18 nonepilepsy controls and 11 epilepsy controls were studied. In 16 cases, representing all three groups, ex vivo 9.4T MRI of the brainstem was carried out. Five regions of interest (ROI) were delineated, including the reticular formation zone (RtZ), and actual and relative volumes (RV), as well as T1, T2, T2* and magnetization transfer ratio (MTR) measurements were evaluated on MRI. On serial sections, actual and RV estimates using Cavalieri stereological method and immunolabelling indices for myelin basic protein, synaptophysin and Microtubule associated protein 2 (MAP2) were carried out in similar ROI. Results: Lower relative RtZ volumes in the rostral medulla but higher actual volumes in the caudal medulla were observed in SUDEP (P < 0.05). No differences between groups for T1, T2, T2* and MTR values in any region was seen but a positive correlation between T1 values and MAP2 labelling index in RtZ (P < 0.05). Significantly lower MAP2 LI were noted in the rostral medulla RtZ in epilepsy cases (P < 0.05). Conclusions: Rostro‐caudal alterations of medullary volume in SUDEP localize with regions containing respiratory regulatory nuclei. They may represent seizure‐related alterations, relevant to the pathophysiology of SUDEP